Many species of microorganism (termed microbes below), particularly including bacteria and single-cell fungi (as yeast or mold), but also algae and protozoa, can be identified mass spectrometrically with a high degree of certainty by breeding a colony in the usual way on a nutrient medium, then transferring small quantities of microbes from the colony to a mass spectrometric sample support plate, and measuring a protein profile directly with a mass—spectrometer. The mass spectrum particularly represents the masses and abundances of the different soluble proteins which are present in sufficiently high concentration in the microbes. This protein profile of the microbes is used to determine their identity by a similarity analysis with reference spectra from a spectrum library.
Specialized mass spectrometers for this purpose and corresponding evaluation and similarity analysis programs are commercially available. At present, this identification procedure proves to be highly successful and rapidly conquers the microbiological laboratories all over the world.
“Identification” of the microbes means categorizing them into the taxonomic hierarchical classification scheme: Domain (eukaryotes and prokaryotes), kingdom, phylum, class, order, family, genus, species and subspecies. The identification of a microbe sample involves determining at least the genus, usually the species, and if possible the subspecies or even the strain as well, which is important, for example, if different subspecies or strains have different pathogenicity. In a more general sense, an identification can also mean a characterization in terms of other, more individual characteristics of the microbes, for example the resistance of a microorganism against antibiotics.
The nutrient medium for the cultivation of a colony is usually contained as an agar in a Petri dish, which normally results in the growth of pure strains in the form of separated microbe colonies in around six hours to some days, depending on the reproductive power of the microbes. If the colonies superimpose or strongly mix, pure colonies can be obtained in a second cultivation, again carried out in the usual way. In a most simple method, some microbes transferred from a selected colony to the mass spectrometric sample support with a small swab are then sprinkled with a strongly acidified solution of a conventional matrix substance (usually α-cyano-4-hydroxy cinnamic acid, HCCA, or 2,5-di-hydroxy-benzoic acid, DHB) for an ionization by matrix-assisted laser desorption (MALDI). The acid (usually formic acid or trifluoroethanoic acid) attacks the cell walls, and the organic solvent (usually acetonitrile) of the matrix solution can penetrate into the microbial cells and cause their weakened cell walls to burst. The sample is then dried by evaporating the solvent, which causes the dissolved matrix material to crystallize. Soluble proteins and, to a much lesser extent, other substances of the cells are embedded into the matrix crystals.
The matrix substance crystals with embedded analyte molecules are then bombarded with focused flashes of UV laser light in a mass spectrometer, creating ions of the analyte molecules in the hot plasma of the vapor cloud; these ions can then be separated according to their mass and measured in the mass spectrometer. Specialized MALDI time-of-flight mass spectrometers (MALDI-TOF-MS) are normally used for this purpose. The mass spectrum is the profile of the mass values of these analyte ions. These are quite predominantly protein ions, the ions with most useful information having masses of between 3,000 daltons and 15,000 daltons approximately. The protein ions in this method are predominantly only singly charged (charge number z=1), which allows for simply referring to the mass m of the ions instead of always using the term “mass-to-charge ratio” m/z, as is actually necessary and conventional with other types of mass spectrometry.
The profile of the proteins is very characteristic of the microbe species in question because each microbe species produces its own set of genetically predetermined proteins each having its own characteristic molecular mass. The abundances of proteins with higher concentrations which can be detected by mass spectrometry, are also widely genetically controlled and depend only slightly on the nutrient conditions or maturity of the colony. The protein profiles are similarly characteristic of the types of microbes as fingerprints are characteristic of individual humans Nowadays, reliable and validated libraries with well documented reference mass spectra of microbes are being thoroughly extended by many laboratories in public and private research institutions and in microbiological institutes of universities. The reference libraries must fulfill strong requirements to be medically and legally admissible.
This method of identification has proven to be extraordinarily successful. The certainty of a correct identification is far greater than that of the microbiological identification methods used until now. It has been possible to prove that the certainty of the identification was way above 95 percent for hundreds of different types of microbe. In most cases of doubt, where there were some deviations from results of microbiological identification methods used until now, genetic sequencing has confirmed the correctness of the mass spectrometric identification.
If the library does not contain a reference mass spectrum for a species of microbe under investigation (which occasionally happens due to the millions of microbe species and the limited size of the current spectral libraries), library searches can usually produce valuable classifications into the higher taxonomic levels of the genus or family of the microbes, because related microbes frequently contain a number of identical types of protein. These cases are increasingly rare, however; pathogenic microbes have now practically all been recorded in the form of reference spectra and can therefore usually be accurately identified down to the level of microbe species.
The method briefly described above of using a small swab to spread some microbes from a colony onto a sample spot of a mass spectrometric sample support which is then sprinkled with a matrix solution, is the simplest and, as yet, fastest type of sample preparation. If a colony is just visible after cultivation, it takes only one to two hours in maximum until the identification is complete even if hundreds of samples have to be analyzed at the same time. Mass spectrometric sample supports with 48, 96 or 384 sample spots each are commercially available; acquisition of mass spectra from these numbers of samples takes around half an hour to two hours. If the identification is urgent, individual microbe samples can be identified in a few minutes (albeit after cultivation, which is always time-consuming)
Other methods of sample preparation have also been investigated, such as extracting the proteins after the microbes have been destroyed by sonication or mechanical treatment, or methods for extracting the proteins from microbes after the cell walls, which are sometimes hard, have been weakened by aggressive acids. These disintegration methods are used when the normal method of swabbing fails because the cell walls of the microbes are not destroyed by sprinkling with the matrix solution. If the swab methods produce mass spectra which are good enough for a comparison, all the disintegration methods provide spectra which are very similar to those of the swab methods, they often even show a lower interfering background in the mass spectra.
Today, mass spectra of the microbe proteins are usually acquired in the linear mode of MALDI time-of-flight mass spectrometers (MALDI-TOF) because these have a particularly high detection sensitivity, even though the mass resolution and the mass accuracy of the spectra from time-of-flight mass spectrometers in reflector mode are much better. In reflector mode, only around one twentieth of the ion signals appear, however, and the detection sensitivity is one or two orders of magnitude worse. The high sensitivity is based on the fact that not only the stable ions but also the much more abundant fragment ions and even the neutral particles from a so-called “metastable” decay which occurs during the flight of the ions, are detected in the linear mode of a time-of-flight mass spectrometer. Secondary electron multipliers (SEM) are used as ion detectors, measuring molecular ions, fragment ions and neutral particles because they all generate secondary electrons on impact. All fragment ions and neutral particles generated after acceleration in the ion source from one species of parent ion have the same speed as the parent ions and thus arrive at the ion detector at the same time. The arrival time is a measure of the mass of the ions which were originally undecomposed.
The increased detection sensitivity is so crucial for many applications that one accepts many of the disadvantages of operating the time-of-flight mass spectrometers in linear mode, such as a significantly lower mass resolution, for example. The energy of the desorbing and ionizing laser is increased for these applications, something which increases the ion yield but also increases their instability, although this is of no consequence here.
Acquiring mass spectra with time-of-flight mass spectrometers generally requires that a very high number of individual spectra are measured and digitized in rapid succession, the individual spectra usually being added together by adding measurement points with the same time-of-flight to form a sum spectrum. The ions for each individual spectrum are generated by one laser flash from a UV pulse laser for each spectrum. The sum spectra have to be generated in this way because of the low dynamic range of measurement and high noise on the signal in the individual spectrum. A minimum of approximately 50, in some cases even 1,000 and more individual spectra are acquired here; a sum spectrum generally consists of several hundred individual spectra which modern mass spectrometers acquire and add together in a few seconds.
For the identification of microbes, usually mass spectra from around 2,000 daltons to high mass ranges of 20,000 daltons are measured. However, mass signals in the lower mass range up to around 3,000 daltons do not have a high degree of reliability because they originate to a large part from coating peptides externally attached to the microbes, from fatty acids depending on kind and availability of nourishment, and a variety of other substances which are present only by chance. The best identification results are obtained if only the mass signals in the mass range between 3,000 and 15,000 daltons are evaluated. The low mass resolution, which occurs for the reasons given, means that the isotope groups whose mass signals each differ by one dalton can no longer be resolved in this mass range. The mass signals, therefore, reflect the shape of the envelopes of the isotope groups.
This method of identifying microbes requires a pure culture of identical microbes, a so-called “isolate”, in order to obtain a mass spectrum which is not superimposed with signals of other types of microbes. It turned out, however, that mass spectra of mixtures of two microbe species can also be evaluated by special methods and that both microbe species can be identified. The identification certainty suffers only slightly. If more than two microbe species contribute to the mass spectrum, the identification probability and identification accuracy decrease very strongly.
The identification of microbes is particularly important for infectious microbes within the blood stream, called septicemia. The microbes usually are released into the blood continuously or in batches from unknown focuses of infections. It is important here that pathogen species are identified very early to commence a targeted medical treatment with correct antibiotics as soon as possible.
The mass spectrometric identification method competes with PCR analysis, where certain genetic sequences of the microbe's DNA, which are characterized by selectively operating pairs of primers, are replicated by polymerase chain reaction (PCR). These methods are fast and can lead to results within a few hours. These PCR analytical methods, however, need some prior knowledge of species, genera, families or classes of the microbes in order to select correct primer pairs. In general, only a “coarse” classification is performed according to the characteristics gram-positive or gram-negative, for example. The determination on the level of a microbe species is only possible in individual cases and requires a very targeted approach based on assumptions. The method is usually limited to individual, frequently occurring and particularly dangerous pathogens such as Staphylococcus aureus, for example. Positive identifications of individual microbe species remain valuable lucky strikes. In cases of negative identification, the knowledge of the exclusion of such dangerous microbes is certainly also valuable, but does not provide a basis for a therapy. The accurate determination of the microbe species must then be left to the conventional microbiological methods, which can quite easily take three to five days, however.
German Patent Application DE 10 2007 058 516 A1 (WPO 2009/065580 A1) discloses a method that directly separates pathogens from body fluids by centrifugation or filtration so that microbes can be transferred from the deposits (centrifugation or filtration pellets) onto the sample support plate. A still better method, also described in the document, is to disintegrate the microbes of the deposits after removal of the supernatant still within the centrifugation tube, for example by adding a few microliters of a strongly acidified matrix solution, with subsequent transfer of the solution with the released proteins onto the mass spectrometric sample support plate. With centrifugation, this disintegration is even possible when no visible pellet is produced. The limit of visibility for a centrifugation pellet is around 106 microbes; the detection limit, in contrast, is currently about 104 microbes, but may be still improved in the future. 104 microbes usually contain more than 100 picograms of soluble proteins, the mass spectrometric detection limit is far below this, however. Since a cultivation stage is not required for infections in clear body fluids, identification in the mass spectrometric laboratory with this method of direct centrifuging of the body fluids can performed within a few minutes.
This method of direct centrifugation or filtration of the microbes is successful because, in the vast majority of cases (far more than 70 percent), acute microbial infections in body fluids are caused by only one single microbe species. At a low percentage of around 15 percent two microbe species are involved, in these cases usually both can be recognized in the mass spectra. This species purity of the pathogens of acute infections is in sharp contrast to other occurrences of microbes in or on the human body—the approximately 1014 bacteria of the intestinal flora in a human intestine comprise at least 400 species of bacteria which live in equilibrium with each other, for example. But the method of direct centrifugation is only successful if these microbes are present in very high concentrations of more than 104 microbes per milliliter which is very rare for internal body fluids. Body fluids are generally sterile, i.e., they usually do not contain any microbes.
As described in the cited document, this method of sedimenting the microbes in a centrifuge or micro-filter can be applied directly and with a high degree of success to all clear body fluids such as lymph, synovial fluid or cerebrospinal fluid (liquor) and even to excreted body fluids such as urine or lachrymal fluid. For body fluids containing endogenous particles, such as whole blood, for example, intermediate steps must be introduced of first growing the microbes by cultivating the blood, because the concentration of microbes are usually only in the range of 0.5 to 10 microbes per milliliter, and second to completely and thoroughly destroy the blood particles such as erythrocytes or leukocytes to thoroughly get rid of all human proteins. In the documents cited, this destruction is exemplarily done by addition of distilled water. The very delicate cell membranes of the human particles are easily destroyed by the osmotic pressure of the water entering, in contrast to the much harder cell walls of bacteria. By repeated addition of distilled water and centrifugation a sufficiently clean pellet should be obtained which should no longer contain any residues of the human particles.
However, experiments carried out in the applicant's laboratory with this method of adding distilled water did not succeed in providing sufficiently clean centrifuge deposits, even when the washing and centrifuging processes were repeated a few times. Even if the deposits did not retain a slightly reddish color, superimpositions with human protein signals always interfered with the microbial protein signals from these deposits to such an extent that an identification becomes uncertain.
There is a need for a fast method for the clean separation of the pathogens of a septicemia in blood, without any traces of remaining human proteins, so that mass spectrometric identification down to the level of species or subspecies and, in addition, other investigations of the pathogens become possible.